Extended access to fentanyl vapor self-administration leads to addiction-like behaviors in mice: Blood chemokine/cytokine levels as potential biomarkers

Abstract

Rodent models are useful for understanding the mechanisms that underlie opioid addiction, but most preclinical studies have focused on rewarding and consummatory aspects of opioids without components of dependence-induced escalation of drug taking or seeking. We characterized several opioid-related behaviors in mice using a model of vaporized fentanyl self-administration. Male and female C57BL/6J mice were assigned to short-access (ShA; 1 h, nondependent) or long-access (LgA; 6 h, dependent) fentanyl vapor self-administration and subsequently tested in a battery of behavioral tests, followed by blood collection during withdrawal. Compared with mice in the ShA group, mice in the LgA group escalated their fentanyl intake, were more motivated to work to obtain the drug, exhibited greater hyperalgesia, and exhibited greater signs of naloxone-precipitated withdrawal. Principal component analysis indicated the emergence of two independent behavioral constructs: "intake/motivation" and "hyperalgesia/punished seeking." In mice in the LgA condition only, "hyperalgesia/punished seeking" was associated with plasma levels of proinflammatory interleukin-17 (IL-17), chemokine (C-C motif) ligand 4 (CCL-4), and tumor necrosis factor α (TNF-α). Overall, the results suggest that extended access to opioids leads to addiction-like behavior, and some constructs that are associated with addiction-like behavior may be associated with levels of the proinflammatory cytokines/chemokines IL-17, TNF-α, and CCL-4 in blood.

Keywords: Addiction-like behavior; Extended access; Hyperalgesia; Operant self-administration; Opioid use disorder (OUD); Punishment; Substance use disorder (SUD).

Fig. 1.. Fentanyl vapor leads to detectable blood fentanyl and norfentanyl levels and antinociception.

(A) Temporal distribution of noncontingent fentanyl vapor deliveries. (B) Concentration of fentanyl and norfentanyl in blood in male and female mice. Eighteen mice were exposed to 1, 3, or 10 fentanyl vapor deliveries (1.5 s, 60 W) and euthanized 2 min after the last vapor exposure for blood collection. Fentanyl and norfentanyl levels were analyzed by liquid chromatography/tandem mass spectroscopy. The data are expressed as individual points and were analyzed by linear regression (n = 6/number of deliveries). (C) Antinociceptive response to fentanyl vapor self-administration. Male and female C57BL/6J mice were tested in the hot plate test (52.5 °C, 30 s cutoff) immediately before and after the fifth 1-h fentanyl vapor self-administration acquisition session. Fentanyl vapor self-administration increased the latency to a nociceptive response. The data are expressed as the mean ± SEM and were analyzed using paired Student’s t -test. ^{∗ ∗ ∗ ∗} p < 0.0001. Antinociceptive responses positively correlated with the number of fentanyl vapor deliveries in the self-administration session. The data are expressed as individual points and were analyzed by linear regression. BL, baseline; ACQ, acquisition (n = 16 mice/sex).

Fig. 2.. Escalation and re-escalation of fentanyl vapor self-administration.

Male and female C57BL/6J mice were trained to lever press for fentanyl vapor deliveries on an FR1 schedule of reinforcement. Upon stable lever pressing, the mice were split in short-access (ShA; 1 h) and long-access (LgA; 6 h) conditions. (A) Mice in the LgA condition escalated their intake in the first hour of the session across days. The data are expressed as the mean ± SEM and were analyzed using two-way repeated-measures ANOVA. ^#p < 0.05, compared with session 1; *p < 0.05, compared with LgA. (B) Mice in the LgA condition escalated their fentanyl intake across the 6 h sessions. The data are expressed as the mean ± SEM and were analyzed using two-way repeated-measures ANOVA. ^#p < 0.05, compared with session 1; *p < 0.05, compared with LgA. (C) Mice in the LgA condition had higher calculated slopes of the escalation curve compared with mice in the ShA condition. The data are expressed as the mean ± SEM and were analyzed using unpaired Student’s t-test. *p < 0.05, compared with ShA. (D) Fentanyl intake on the first re-escalation self-administration sessions. The data are expressed as the mean ± SEM and were analyzed using two-way repeated-measures ANOVA. ^#p < 0.05, compared with session 1. (E) Mice in the LgA condition maintained higher fentanyl intake across sessions in the re-escalation phase. The data are expressed as the mean ± SEM and were analyzed using two-way repeated-measures ANOVA. ^# p < 0.05, compared with session 1; *p < 0.05, compared with LgA. (F) The calculated re-escalation slope did not differ between ShA and LgA conditions. The data are expressed as the mean ± SEM and were analyzed using unpaired Student’s t-test. (n = 8/sex/group).

Fig. 3.. Naloxone-induced signs of withdrawal, hyperalgesia during spontaneous withdrawal, and motivation for fentanyl.

After escalation, male and female C57BL/6J mice were tested in a battery of tests to assess somatic and motivational signs of withdrawal. (A) Naloxone-precipitated withdrawal. Immediately after fentanyl vapor self-administration session 10, all mice received naloxone (1 mg/kg, IP) and were observed for 20 min for signs of withdrawal. The data are expressed as the mean ± SEM and were analyzed using unpaired Student t-test. ^**p < 0.01, compared with ShA. (B) Mechanical hyperalgesia. Between 36 to 40 h after a self-administration session (i.e., during spontaneous withdrawal), the mice were tested for mechanical hyperalgesia using an electronic von Frey device. The data are expressed as the mean ± SEM and were analyzed using unpaired Student t-test. *p < 0.05, compared with ShA. The dotted line represents the average baseline measure (i.e., before fentanyl exposure) for all mice; both groups developed hyperalgesia compared with the baseline (BL) measure. (C) Progressive ratio test (motivation or”effort”). After escalation, all mice were tested in a progressive-ratio task, in which the number of lever presses that were required for the next fentanyl delivery increased by 6. The data are expressed as the mean ± SEM and were analyzed using unpaired Student’s t-test. *p < 0.05, compared with ShA. (D) Time delay task (motivation). After escalation, all mice were tested in the delayed-reward task, in which the time between lever presses and vapor delivery increased by 6 s. The data are expressed as the mean ± SEM and were analyzed using unpaired Student’s t-test. *p < 0.05, compared with ShA (n = 8/sex/group).

Fig. 4.. Self-administration despite punishment.

Male and female C57BL/6J mice were tested for vapor self-administration in 1 h-sessions with vehicle or fentanyl that was adulterated with increasing concentrations of capsaicin. (A) Vapor deliveries for each concentration of capsaicin alone in vehicle without fentanyl. The data are expressed as the mean ± SEM and were analyzed using two-way repeated-measures ANOVA. ^#p < 0.05, compared with 0% regardless of group. (B) Vapor deliveries for each concentration of capsaicin in fentanyl. The data are expressed as the mean ± SEM and were analyzed using two-way repeated-measures ANOVA. ^#p < 0.05, compared with 0% (n = 8/sex/group).

Fig. 5.. Principal component analysis.

The behavioral measures were analyzed separately for each group (ShA and LgA) by a Varimax-normalized PCA. Factor loadings ≥ 0.6 were considered. Black symbols represent all measures that loaded on factor 1 (x-axis). Orange circles represent measures that loaded on factor 2 (y-axis). The blue circle represents the only measure that loaded on factor 3. SWD, precipitated (somatic) withdrawal; PR, progressive ratio; vF, von Frey mechanical nociception (n = 8/sex/group).

Fig. 6.. Correlation analysis.

Individual factor loadings from the PCA were correlated with plasma levels of cytokines and corticosterone in ShA (A) and LgA (B) groups. Data were analyzed by Pearson’s correlations using two-tailed p values with α= 1%. The p values are represented inside the boxes. Orange squares denote p < 0.05–0.001. Green squares denote p < 0.10. (n = 7–8/sex/group).